This application focuses on identifying molecular mechanisms by which an emerging tumor suppressor sphingolipid ceramide regulates the expression of human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase that maintains/elongates telomeres at the end of chromosomes, and plays important roles in the pathogenesis of lung and other cancers. On the other hand, ceramide is an established bio-effector sphingolipid, which mediates anti-proliferative biological responses, such as senescence, growth arrest, or apoptosis. Recent studies suggest that hTERT is important in the regulation of cancer cell growth and/or survival, independently of its function in the maintenance of telomeres without affecting net telomere length. Therefore, determining mechanisms that regulate hTERT expression is very important for both cancer research and therapy. In parallel with these data, our studies have revealed that ceramide is one of the upstream regulators of telomerase mediating the inhibition of hTERT expression in human lung cancer cells. The data showed that modulation of hTERT expression by ceramide is mechanistically linked to inactivation of its promoter via Sp3- and c-Myc-dependent pathways, involving their deacetylation or ubiquitination, respectively. Collectively, experimental evidence suggested a central hypothesis that ceramide mediates the inhibition of the hTERT transcription by Sp3- and c-Myc-dependent mechanisms, via their deacetylation and dephosphorylation-ubiquitination, coupled to the regulation of histone deacetylase 1 (HDAC1) and protein phosphatase 2A (PP2A), respectively. To test this hypothesis, two Specific Aims are proposed: Specific Aim 1. Determine the role and mechanism of action of ceramide in Sp3-mediated regulation of hTERT expression;and Specific Aim 2. Define the mechanisms by which ceramide prevents c-Myc-dependent activation of hTERT expression via regulation of its ubiquitination/degradation. These studies will define the mechanisms of ceramide-mediated repression of hTERT expression in the context of HDAC1 function, Sp3-acetylation, and the hTERT promoter network. In addition, they will help understand how ceramide regulates the function of PP2A directly, and will help dissect ceramide-mediated signaling that controls the hTERT promoter by c-Myc in human lung cancer compared to non-cancerous lung fibroblasts and normal bronchial epithelial cells. In the long-term, studies proposed in this application will help develop mechanism-based therapeutic strategies for the treatment of human lung cancer, which is the leading cause of cancer-related deaths in South Carolina and in the United States.